Ecological and Evolutionary Physiology of the Stress Response and Stress Proteins

广泛的目标：拟议的活动继续一项研究计划，其目标是了解复杂的生理和生化机制的起源和维持，使有机体能够在具有挑战性的环境中坚持下去。这个程序使用热休克蛋白Hsp70作为模型系统，Hsp70可以最大限度地减少压力对其他蛋白质的损害，以及果蝇中编码Hsp70的基因。尚未回答的主要问题是：热休克蛋白70水平在自然群体中的差异的遗传基础是什么，这种差异是如何产生的？先前的工作表明，转座元件，即可以从一个基因移动到另一个基因的DNA序列，是这些问题的答案。这项研究的主要目的是验证这一假说。具体目标：首先，研究将确定转座元件实际插入到编码热激蛋白的基因中的频率，并将该频率与在结构、组织和调控方面与热激基因相似或不相似的其他基因的频率进行比较。在这样做的过程中，研究将发现许多转座元件自然插入到热激基因中的例子。接下来，研究将使用这些天然突变体来检查这些插入片段是否真的对热休克基因有预期的影响：减少信使RNA、热休克蛋白和承受压力的能力。最后，研究人员将进行“实验室进化”，即果蝇品系在应该有利于保留或消除其热休克基因中有或没有转座元件的个体的环境中竞争。这项最后的实验将测试热休克基因中的转座元件是否真的对个体适应性具有预期的影响。方法：(A)生物信息学，对先前存在的信息进行分析，以预测基因的性质。(B)高通量筛选，即同时快速分析许多种群中的许多基因，以检测遗传事件。这将使用一种克隆基因的新技术--“通用快步行走”。(C)转录图谱，即特定基因在表达时产生的信使RNA的量化。这种分析将使用两种技术，一种是热休克基因的调节区与报告蛋白的融合，当基因表达时，报告蛋白会发光；另一种是定量实时聚合酶链式反应，它通过量化信使RNA在化学反应中的积累来测量信使RNA。(D)实验室进化，如上所述。意义：拟议的研究具有独特之处，因为它将阐明一种显然反复导致蛋白质表达减少的机制，并产生适应性后果。由于对相关基因/蛋白质的广泛背景工作，它可以严格地将意义赋予调节性突变和它们调节的蛋白质水平的变异。这项拟议的研究有可能成为一项里程碑式的研究，其中解释了变异的进化生成、基因序列、细胞功能、组织功能、适应度、种群中的基因频率以及对自然选择的反应之间经常缺失的所有联系。

Broad goals: The proposed activity continues a research program whose goal is to understand the origin and maintenance of complex physiological and biochemical mechanisms that enable organisms to persist in challenging environments. This program uses as a model system the heat-shock protein Hsp70, which minimizes stress-induced damage of other proteins, and the genes that encode it in the fruit fly, Drosophila. The principal remaining unanswered questions are: What is the genetic basis for the variation in Hsp70 levels among natural populations, and how does this variation arise? Prior work suggests that transposable elements, DNA sequences that can move from gene to gene, are the answer to these questions. The major goal of the research is to test this hypothesis.Specific aims: First the research will determine the frequency with which transposable elements actually insert in the genes encoding heat-shock proteins, and compare this frequency with that for other genes that either do or do not resemble heat-shock genes in their structure, organization, and regulation. In so doing, the research will discover numerous instances of transposable elements naturally inserting into heat-shock genes. Next, the research will use these natural mutants to examine whether the insertions actually have the expected effect on heat-shock genes: reduction in messenger RNA, heat-shock protein, and ability to withstand stress. Finally, the researchers will undertake "laboratory evolution", in which fruit fly strains compete in environments that ought to favor retention or elimination of individuals with and without transposable elements in their heat-shock genes. This final experiment would test whether transposable elements in the heat-shock genes actually have their expected effect on individual fitness.Approaches: (a) Bioinformatics, the analysis of pre-existing information to predict genes' properties. (b) High-throughput screening, the simultaneous and rapid analysis of many genes in many populations to detect genetic events. This will use "universal fast walking", a new technique for cloning genes. (c) Transcriptional profiling, the quantification of messenger RNAs produced by specific genes when they are expressed. This profiling would use two techniques, the fusion of the heat-shock genes' regulatory region with a reporter protein that emits light when the gene is expressed, and quantitative real-time PCR, which measures messenger RNA by quantifying its accumulation in a chemical reaction. (d) Laboratory evolution, as described above.Significance: The proposed research is distinctive in that it will elucidate a mechanism that apparently has repeatedly resulted in the decreased expression of a protein, with adaptive consequences. Because of extensive background work on the genes/protein in question, it can rigorously assign significance to both regulatory mutations and variation in the level of the protein they regulate. The proposed research has the potential to become a landmark study in which all of the often-missing links among evolutionary generation of variation, gene sequence, cellular function, organismal function, fitness, gene frequency in populations, and response to natural selection are explained.